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Modulation of the Immune System: Treatment Options and new Developments PD Dr. Chris Rundfeldt DrugConsult.Net Magdeburg and Dresden, Germany Diseases and conditions with (pathological) involvement of the immune system Inflammatory conditions, associated with trauma, infection or tissue destruction Chronic perpetuating inflammations: COPD, arthritis Allergic diseases: type I-IV Atopic diseases: atopic dermatitis, atopic rhinitis, asthma Autoimmune related diseases: rheumatoid arthritis, inflammatory bowl disease, colitis ulcerosa, morbus Crohn, psoriasis, multiple sclerosis, coeliac disease, Rasmussen's encephalitis, myasthenia gravis, …… Organ / tissue transplantation and rejection reactions Localized effects of the ubiquitous organ “Immune system” The immune system is involved in numerous physiological and pathological processes Each organ can be a disease target (CNS, skin, gut, muscle, soft tissue…..) Often targeted tissues are limited and reasons for localized effects are not known Potential reasons for localization are hidden in the complex nature of such multifactor diseases A drug with immune modulator activity can be useful for diverse diseases For example glucocorticoids are used for many conditions Intervention options: well established drugs Anti-inflammatory drugs Immune modulators Inhibitors of cyclooxygenases (Ibuprofen...) Inhibitors of lipoxygenases (Zileuton…) Leukotriene receptor antagonists ( Montelukast…) Glucocorticoids Selective cytokine blockers (Biologics) TNFa inhibitor antibody / fusion protein (Infliximab, Ethernacept…) IL-1 receptor antagonist (Ankarinra) Anti-T-cell fusion protein (Alefacept) ……. Intervention options: Immunosuppressives Antimetabolites Calcineurin inhibitors (IL2 synthesis inhibitors) Cyclosporin Tacrolimus, Pimecrolimus mTOR, kinase inhibitor, activation of T-cells inhibited Purine synthesis inhibitors (Azathioprine …) Purine analog mimics adenosine, DNA synth. Inhibit. (Cladribine) Pyrimidine synthesis inhibitors (Leflunomide …) Antifolate (Methotrexate) Sirolimus (=Rapamycin) TNFa-synthesis inhibitor (Thalidomide, Lenalidomide) (Thalidomide: teratogenic effect phocomelia, now used for Erythema nodosum leprosum, cancer, etc. Limitations of pharmacological intervention Immune suppressive agents can facilitate potentially life threatening infections Invasive aspergillosis Tuberculosis General infections Unexpected adverse events include disturbance of learning and memory (Rapamycin) Inhibition of rapidly dividing tissues Glucocorticoids lack a good separation of pharmacologically active and toxic doses Limitations of pharmacological intervention: Biologics Biologics are thought to be selective but can induce: Flu-like symptoms, nausea, fatigue, loss of appetite Redness, rash, and/or pain at injection site Allergic reaction to mouse protein with monoclonal antibodies Increased risk of Hodgkin’s and non-Hodgkin’s lymphoma and other types of cancer in children and teens taking TNF inhibitors Lupus-like syndrome Possible reactivation of latent tuberculosis infections with TNF inhibitors Progressive Multifocal Leukoencephalopathy (PML), see Natalizumab case (Tysabri) Induction of antibodies against therapeutics results in loss of effect New treatment options? Novel treatment options should be: Selective for pathological conditions (reduce overactivation of the immune system component) Have a good separation of effects and adverse reactions Be easy to handle, at best oral treatment While no such drugs are readily available, a few interesting candidates can be discussed Examples for new treatment options Inhibitors Stabilized of Phosphodiesterase 4 cAMP analogues Dimethylfumarate Raft-modulators Phosphodiesterase 4 inhibitors Inhibitors of Phosphodiesterase 4 (PDE4) Prototype: Rolipram Initial development as antidepressant Early investigation in multiple sclerosis indicate pharmacological effect, but GI related side effects Numerous development projects worldwide for diverse diseases Currently only roflumilast marketed in EU for treatment of COPD Other potential indications for roflumilast: Asthma, atopic dermatitis, psoriasis Roflumilast (PDE4i) Initial studies in patients with asthma Roflumilast (PDE4i) Significant effects in patients with COPD But effect size lower than known for Theophyllin! Roflumilast (PDE4i) Significant effects: reduction of exacerbations Novel PDE4i: how to test preclinically In vitro tests: Cytokine release in PBMCs or whole blood Induction with LPS or anti-CD3/CD28 or similar Ex vivo tests: Cytokine release from whole blood In vivo tests: Ovalbumin induced eosinophilia in sensitized BN rats (asthma model) LPS-induced neutrophilia in Lewis rats and in ferrets (COPD model) Emesis in ferrets and domestic pigs Example development strategy for PDE4i Quality Lead Metabolic stability in plasma and liver prepar., species comparison, metabolite profile Stable in plasma, no or low turnover in liver preps. Expl. kinetics in rat and dog (i.v. vs p.o.) Dog: t 1/2 > 2 h BV > 30% Inhibition of cytokine release - PBMC‘s ; Maph (IL2, 4, 5, 13, IFN) (IL1, 6, 8, 12, 15, 17, MCP1) IC50 < 100 nM LTB4 (neutrophils) LTC (eosinophils) IC50 < 100 nM CYP3A4 Induction Effect on- LPS-induced neutrophilia in rats and ferrets - late phase eosinophilia in rats - bronchial hyperreactivity in mice Equal or better than Roflumilast Side effect profile in - ferrets (emesis and CNS) - pigs (cardiovascular) - mice, rats (CNS) TI > 10 no induction Preselection preclinical candidate Example development strategy for PDE4i Genotoxicity - Ames test - chromosome aberration test - micronucleus test No effect Receptor screening initiated initiated Selectivity > 100 Preclinical Candidate PK / ME Identification of - major metabolites - metabolic pathways - involved enzymes Safety Efficacy Tolerability - Irwin test (rat) - acute toxicity (rat, mice, dog) Respiratory models - allergic rhinitis (rat) - LPS-induced neutrophilia ( ferrets and pigs) - allergen-induced bronchoconstriction (guinea pigs) acceptable 14C Pharmacokinetic and metabolic profiling Full 4-week tox package Clinical Program Non-respiratory inflammation models - skin (mice) - other models (CRO) CNS diseases Indication extension Example data: Ovalbumin induced eosinophilia Novel compound Example data: LPS induced neutrophilia Novel compound Example data: Safety profile in experimenta animals Rationale: clinical dose of roflumilast is limited by CNS side effects, not by emesis Test Novel Roflumilast LPS neutrophilia >50% 0.01 mg/kg 0.01 mg/kg Highest dose tested without emesis 3 mg/kg 0.1 mg/kg Highest dose tested without CNS effects 0.3 mg/kg 0.01 mg/kg not free of CNS effects TI in relation to emesis p.o. ~300 ~10 TI in relation to CNS effects p.o. < 1, no separation ~30 Compounds were administered to ferrets with gastric tube as a solution or suspension Translation of preclinical data in phase I Phase I is dedicated to determination of safety Options to get insight in efficacy in phase I LPS induced TNFa release from whole blood i.v. LPS induced hyperthermia and flu-like reaction in volunteers Intratracheal LPS administration followed by lung lobe lavage Local non-allergic or allergic skin irritation in sensitive patients, with skin prick test or with Balsam of Peru Allergen induced late phase bronchoconstriction in volunteerpatients with mild stable asthma Methacholine induced bronchoconstriction Local non-allergic or allergic skin irritation in sensitive patients, with skin prick test or with Balsam of Peru PDE4 inhibition, other options Increased cAMP levels: cAMP substitution instead of PDE4 inhibition Stable cAMP analogues can be active but activate also protein kinases Clinically known stable analogue: Dibuturyl-cAMP (bucladesine) Bucladesine is a safe drug, no emesis Was developed as cardiotonic for i.v. administration Was developed as skin healing cream Failed for different reasons: Cardiotonic function difficult to test, skin cream has strong odor (butyric acid is released) Clinical data showing a homogenous clinical effect of cAMP level modulation Goyarts et al., Skin Pharmacol Appl Skin Physiol 2000:13, 86-92 Increase in thickness (mm) DB-cAMP, water free ointment 0.15 0.10 ** 0.05 0.00 vehicle 0.5% 1.5% Increase in thickness (mm) Dibuturyl-cAMP is active in arachidonic acid induced ear swelling in mice 0.15 Ketoprofen, commercial cream 0.10 0.05 *** 0.00 vehicle ketoprofen DB-cAMP Drugs were administered onto the outer surface of the mouse ear 3 h before arachidonic acid challenge. Measurement of ear thickness. 60 min later the ear thickness was measured. 2-deoxy-cAMP may be an interesting candidate for a topical antiinflammatory ointment, but safety of this compound is not tested No patent protection possible Fumaric acid esters: old drugs with novel mode of action Dimethyl fumarate: A novel old drug for oral treatment of immune diseases Dimethyl fumarate (DMF) is marketed in Germany as main component of a first line oral drug for treatment of moderate to severe psoriasis (Fumaderm®) Fumaric acid esters are an effective therapy in patient with moderate to severe psoriasis, even in those who have previously been intolerant of systemic therapy The mode of action of DMF is not fully understood Problem: tolerability, GI irritation and flush, skin irritation Problem: Papilloma / carcinoma in forestomach rats Biogen Idec has developed a novel micro-tablet formulation (immediate release) and tested positive for psoriasis and MS. Now in phase III for MS Forward Pharma has solved the challenge of developing a modified release formulation. Now in phase II for Psoriasis Fumaric acid ester (FAE) and Dimethylfumarate: Pharmacology FAEs cause an increase of the intracellular calcium concentration FAEs inhibit the expression of the ICAM-1 T-cell receptor ligands The treatment of psoriatic patients with Fumaderm® results in a reduced infiltration of the skin with granulocytes and T helper cells, followed by a reduction of the acanthosis and hyperkeratosis Monomethyl fumarate increases the IL-4 and IL-5 production of activated T cells, in a dose-dependent manner Dimethyl fumarate inhibits cytokine-induced E-selectin, VCAM-1 expression in human endothelial cells Dimethyl fumarate inhibits cytokine-induced nuclear translocation of NF-kB. Dimethyl fumarate induces apoptosis in human monocyte-derived dendritic cells. Monomethyl fumarate suppresses the FMLP-stimulated respiratory burst in granulocytes Fumaric acid ester (FAE) and Dimethylfumarate: Pharmacology (2) In in vitro tests DMF is more potent than MMF In plasma samples, only MMF can be found (rapid demethylation) In urine, DMF-conjugates with glutathione were found Proposed MOA: Intracellular uptake in leukocytes Rapid conjugation with glutathione Dimethyl fumarate induces immune suppression via glutathione depletion and subsequent induction of heme oxigenase DMF pathways Lumen -Gut wall- ERY Tablet DMF Blood streem ERY no metabol. ERY ERY 1 DMF Plasma Esterases MMF 2 WBC: DMF DMF-GSH Active Transp. ERY DMF-GSH WBC: MMF MMF-GSH Active Transp. Plasma Esterases MMF-GSH Mercapturic acid derivatives, excreted in urine Glutathione depletion is immune suppressive Top: PBMCs were stimulated with LPS/IFNg (1 mg/ml, 10 ng/ml) or PHA (100 mg/ml) in the presence of DMF Bottom: Mixed lymphocyte reaction to quantify dose response and reversibility Substitution of reduced GSH abolishes the inhibition of inflammatory cytokine secretion and alloreactive lymphocyte proliferation by DMF and DEF FAEs are active in moderate to severe psoriasis Mrowietz et al. Br. J. Dermatol. 1998 138: 456-460 Effect of DMF in MS patients (Biogen) Tabel from: Lee et al., The International MS Journal 2008; 15: 12–18 Figure from: Kappos et al., Lancet 2008; 372: 1463–72 Raft modulation as novel lipid chemistry target Raft modulation: Innovation ahead? Lipid Rafts are specific clusters of lipids and proteins regulating a wide range of biological and pathological processes Lipid Rafts concentrate proteins to increase process efficacy Rafts coordinate disease events at specific sites and times Signal transduction, virus entry & budding, bacterial entry, receptor recycling, protein maturation are dependent on Lipid Rafts Each process occurs in a Raft of unique composition Rafts are novel pharmaceutical targets Example: IgE receptor RAFT as drug target IgE-receptor clustering drives RAFT formation Protein-driven (specificity) reaction is combined with novel lipid chemistry target Receptor clustering and RAFT coalescence IgE FceRI multivalent allergen RAFT lipids kinase unrelated RAFT receptor Signal cascade, release of immune modulators Miltefosine is a clinically effective RAFT modulator Skin-prick test on human volunteers; IgE-dependent allergic response Pretreatment with miltefosine (Miltex) or saline Skin-prick with allergen & positive control (histamine) Weller et al. 2008 Miltefosine inhibits human mast cell activation in-vitro and in-vivo. J Invest Dermatol ifl D or as on e 0 6% 500 ilt ef . 1000 M *** 2% *** ilt ef . 1000 M *** ilt M ilt M D 6% 2% ifl or ef . ef . to p on tr ol 0 Ve hi cl e 2000 ear swelling (µm) *** Ve hi cl e C m g/ kg m g/ kg *** IL-1 (pg/mg) 5 0 g/ kg m *** on tr ol *** so ne 10 ex a ef . D M ilt 50 lc et ha m g/ kg m g/ kg m 20 sy s on tr ol eh ic le ilt ef . M V C 100 as a ex a 00 20 ef .1 ef . eh ic le on tr ol V lc ear swelling (µm) 150 B D ilt M ilt as a IL-1 (pg/mg) 3000 M B Clinical effect back-translated in preclinical model: TDI induced ear swelling in mice 150 100 * ** 50 *** *** 0 2000 1500 ** * *** *** 0 Conclusion Immune modulator drugs represent a diverse family of drugs Despite the large number of marketed drugs, there is need for better drugs Even among old drugs there are interesting new approaches to immune modulation Recent data indicate the reduction in glutathione concentration can induce immune modulation Raft modulation is a 2nd novel approach to immune modulation Thank you for your attention I am ready to take your questions